Synthesis of three-dimensional calcium carbonate nanofibrous structure from eggshell using femtosecond laser ablation
© Tavangar et al; licensee BioMed Central Ltd. 2011
Received: 11 November 2010
Accepted: 20 January 2011
Published: 20 January 2011
Natural biomaterials from bone-like minerals derived from avian eggshells have been considered as promising bone substitutes owing to their biodegradability, abundance, and lower price in comparison with synthetic biomaterials. However, cell adhesion to bulk biomaterials is poor and surface modifications are required to improve biomaterial-cell interaction. Three-dimensional (3D) nanostructures are preferred to act as growth support platforms for bone and stem cells. Although there have been several studies on generating nanoparticles from eggshells, no research has been reported on synthesizing 3D nanofibrous structures.
In this study, we propose a novel technique to synthesize 3D calcium carbonate interwoven nanofibrous platforms from eggshells using high repetition femtosecond laser irradiation. The eggshell waste is value engineered to calcium carbonate nanofibrous layer in a single step under ambient conditions. Our striking results demonstrate that by controlling the laser pulse repetition, nanostructures with different nanofiber density can be achieved. This approach presents an important step towards synthesizing 3D interwoven nanofibrous platforms from natural biomaterials.
The synthesized 3D nanofibrous structures can promote biomaterial interfacial properties to improve cell-platform surface interaction and develop new functional biomaterials for a variety of biomedical applications.
Autogenous bone has long been considered the ideal grafting material in bone reconstructive surgery owing to its osteogenic, osteoinductive and osteoconductive properties [1, 2]. However, harvesting the autogenous bone requires an additional surgery which increases morbidity at the donor site and extends the operation period [3, 4]. Therefore, a variety of new bone grafting materials has substituted for autogenous grafts thanks to recent advances in biotechnology. Among them, natural bone substitute biomaterials from bovine sources and bone-like minerals (calcium carbonate) derived from corals or avian eggshells, have been preferred due to their biodegradability, abundance and lower price in comparison with synthetic biomaterials [5–9]. The coralline calcium carbonate (calcite), which is totally resorbable and biocompatible and shows good osteoconductivity, has been used as an effective bone substitute in the natural form or converted to hydroxyapatite (HA) in bone healing in dentistry and orthopedic [4, 10–14].
Avian eggshell, with a mineral composition similar to corals, has been introduced as a potential bone substitute in maxillodacial and craniofacial surgery as they could easily be acquired and contain ions of Sr and F[4, 15] and . One of the crucial characteristics to be considered when using a bone substitute graft is its degradation rate due to the fact that it may have effects on the long-term results. The graft should undergo only minimal resorption if it is used as an onlay graft whereas a resorbable one is desirable when a bone substitute is used as interpositional graft or in a peri-implant defect . Eggshell, which can be manufactured under powdered or block form, can be used for both indications.
Many in vitro and in vivo studies have shown that the microporous surface structure and biodegradability of bone substitutes play critical roles in bone healing. It is indicated that cell attachment and proliferation are improved on nanostructure surface than microstructure one .
Among the nanoscale structures, randomly interwoven nanofibrous structures are particularly preferred for scaffolding systems in comparison with nanoparticles due to their continuous structure. The vantage of a surface comprised of ultra-fine, continuous nanofibers would be high porosity, high surface volume ratio, variable pore-size distribution, and first and foremost, morphological similarity to natural Extra Cellular Matrix (ECM) . There are reported studies where eggshell has been used to compose different Ca-precursor nanoparticles or HA nano-powder that requires the additional step such as sintering to synthesize porous surfaces [16, 19]. Whereas, no studies on synthesizing 3D nanofibrous structure on natural biomaterials have been accounted so far. Therefore, a simple method to generate 3D nanofibrous structure in a single-step would be in a great interest.
In the presented work, we have proposed a novel technique to synthesize calcium carbonate 3D nanofibrous structures from eggshells using femtosecond laser processing. To the best of authors' knowledge, this is the first work on synthesizing 3D calcium carbonate nanofibrous structures using femtosecond laser. We also have investigated the effects of laser pulse repetition on the density of nanofibers and the structure pore size.
Results and discussion
Previous in vitro and in vivo studies have pointed out that the microporosity of the bone substitute surface structure as well as its biodegradability play an important role in bone healing. Thus, the generated nanofibrous structure with different porosity shows a different degree of biodegradability when implanted in the biological environment. Microporosity influences the bone substitute dissolution rate in biological fluids; hence a surface with higher porosity shows better degradability. Biodegradation of bone substitutes is vital to initiate the bone deposition process [22, 23]. Porous structures increase adsorption of proteins such as bone morphogenetic proteins and other necessary ones required for bone formation which consequently influences cell adhesion and the subsequent cell proliferation and differentiation of osteoblasts [4, 22, 23]. On the other hand, cell attachment and proliferation are improved for nanostructures in comparison with micron-structures owing to higher effective surface area of the nanofibers . As a result, we believe that the calcite nanofibrous structure generated on the eggshell substrate could enhance the biodegradability as well as the osteoconductivity of the surface in comparison with nanoparticles or micron-structure.
This study describes a novel technique to synthesize calcium carbonate nanofibrous structure from eggshell using high repetition femtosecond laser under ambient condition. To the best of our knowledge, this is the first time that synthesizing 3D calcium carbonate nanofibrous structures using femtosecond laser have been reported. The morphological analyses by SEM and TEM were confirmed that fabricated nanofibers have approximately uniform 3D structure with average size of 50 nm. Further experiments showed that by changing the laser pulse repetition, different nanofibrous structure with different porosity could be achieved. The XRD and EDX analyses showed that laser irradiation barely affects chemical decomposition, though; part of the organic matter believes to be changed to calcium hydroxide owing to laser irradiation. This proposed method suggests a promising step in synthesizing interwoven 3D platforms from natural biomaterials to support new bone formation and achieve rapid bone healing as well as to improve develop new functional biomaterials for various biomedical applications. In vitro test to investigate the degradation rate of the nanofibrous scaffold in physiological environments and cell culture assays to understand the scaffold-cell interaction are being undertaken.
Materials and methods
The avian eggshell representing 11% of the total weight of the egg consists mainly of calcium carbonate (94%), calcium phosphate (1%), organic matter (4%) and magnesium carbonate (1%) . Hen's eggs were purchased, emptied and washed thoroughly with distilled water to get rid of dirt and organic layer.
The nanofibrous structures were then characterized using Scanning Electronic Microscopy (SEM) followed by Energy dispersive X-ray spectroscopy (EDS) analyses. The nanoparticle aggregation and nanofiber size were analyzed by Transmission Electron Microscope (TEM). The samples were sonicated in isopropanol solution to separate the nanostructures from the substrate. Then a drop of the nanofiber-dispersed solution was placed on the copper grid and allowed to dry in a desiccator.
Phase analysis of the synthesized structures was performed using X-ray Diffraction (XRD). The x-ray source was a Cu rotating anode generator (Rigaku) with parallel focused beam and 3-circle diffractometer (Bruker D8) with a 2D detector (Bruker Smart 6000 CCD). The average wavelength of the x-rays was 1.54184Å. Phi scans with widths of 60°were done with the detector at four different swing angles for each sample in order to get a profile with a 2θ range of 10.5-104°.
This research is funded by Natural Science and Engineering Research Council of Canada.
- Hjorting-Hansen E: Bone grafting to the jaws with special reference to reconstructive preprosthetic surgery, a historical review. Oral and Maxillofacial Surgery. 2002, 6: 6-14.Google Scholar
- Park JW, Jang JH, Bae SR, An CH, Suh JY: Bone formation with various bone graft substitutes in critical-sized rat calvarial defect. Clinical Oral Implants Research. 2009, 20 (4): 372-378. 10.1111/j.1600-0501.2008.01602.x.View ArticleGoogle Scholar
- Meijndert L, Raghoebar GM, Schüpbach P, Meijer HJ, Vissink A: Bone quality at the implant site after reconstruction of a local defect of the maxillary anterior ridge with chin bone or deproteinised cancellous bovine bone. Int J Oral Maxillofac Surg. 2005, 34 (8): 877-84. 10.1016/j.ijom.2005.04.017.View ArticleGoogle Scholar
- Park JW, Bae SR, Suh JY, Lee DH, Kim SH, Kim H, Lee CS: Evaluation of bone healing with eggshell-derived bone graft substitutes in rat calvaria: A pilot study. Journal of Biomedical Materials Research Part A. 2007, 87 (1): 203-214.Google Scholar
- Durmus E, Ilhami C, Aydın MF, Yıldırım G, Sur E: Evaluation of the Biocompatibility and Osteoproductive Activity of Ostrich Eggshell Powder in Experimentally Induced Calvarial Defects in Rabbits. Journal of Biomedical Materials Research Part B: Applied Biomaterials. 2007, 86B (1): 82-89. 10.1002/jbm.b.30990.View ArticleGoogle Scholar
- Siva Rama Krishna D, Siddharthan A, Seshadri SK, Sampath Kumar TS: A novel route for synthesis of nanocrystalline hydroxyapatite from eggshell waste. Journal of Materials Science: Materials in Medicine. 2007, 18 (9): 1735-1743. 10.1007/s10856-007-3069-7.Google Scholar
- Vago R, Plotquin D, Bunin A, Sinelnikov I, Atar D, Itzhak D: Hard tissue remodeling using biofabricated coralline biomaterials. J. Biochem. Biophys. Methods. 2002, 50: 253-259. 10.1016/S0165-022X(01)00235-4.View ArticleGoogle Scholar
- Haque S, Rehman I, Darr JA: Synthesis and Characterization of Grafted Nanohydroxyapatites Using Functionalized Surface Agents. Langmuir. 2007, 23: 6671-6676. 10.1021/la063517i.View ArticleGoogle Scholar
- Liang X, Xiang J, Zhang F, Xing L, Song B, Chen S: Fabrication of Hierarchical CaCO3 Mesoporous Spheres: Particle-Mediated Self-Organization Induced by Biphase Interfaces and SAMs. Langmuir. 2010, 26 (8): 5882-5888. 10.1021/la9037815.View ArticleGoogle Scholar
- Yukna RA, Yukna CN: A 5-year follow-up of 16 patients treated with coralline calcium carbonate (Biocoral) bone replacement grafts in infrabony defects. J Clin Periodontol. 1998, 25: 1036-1040. 10.1111/j.1600-051X.1998.tb02410.x.View ArticleGoogle Scholar
- Velich N, Nemeth Z, Toth C, Szabo G: Long-term results with different bone substitutes used for sinus floor elevation. Journal of Craniofacial Surgery. 2004, 15: 38-41. 10.1097/00001665-200401000-00013.View ArticleGoogle Scholar
- Coughlin MJ, Grimes JS, Kennedy MP: Coralline hydroxyapatite bone graft substitute in hindfoot surgery. Foot and Ankle International. 2006, 27: 19-22.Google Scholar
- Vuola J, Goransson H, Bohling T, Asko-Seljavaara S: Bone marrow induced osteogenesis in hydroxyapatite and calcium carbonate implants. Biomaterials. 1996, 17: 1761-6. 10.1016/0142-9612(95)00351-7.View ArticleGoogle Scholar
- Liao H, Mutvei H, Sjostrom M, Hammarstrom L, Li J: Tissue responses to natural aragonite (Margaritifera shell) implants in vivo. Biomaterials. 2000, 21: 457-68. 10.1016/S0142-9612(99)00184-2.View ArticleGoogle Scholar
- Dupoirieux L, Pourquier D, Neves M, Teot L: Resorption kinetics of eggshell: An in vivo study. J Craniofac Surg. 2001, 12: 53-58. 10.1097/00001665-200101000-00009.View ArticleGoogle Scholar
- Siddharthan A, Sampath Kumar TS, Seshadri SK: Synthesis and characterization of nanocrystalline apatites from eggshells at different Ca/ P ratios. Biomed Mater. 2009, 4: 045010-045019. 10.1088/1748-6041/4/4/045010.View ArticleGoogle Scholar
- Fujiharaa K, Kotakib M, Ramakrishna S: Guided bone regeneration membrane made of polycaprolactone/calcium carbonate composite nano-fibers. Biomaterials. 2005, 26: 4139-4147. 10.1016/j.biomaterials.2004.09.014.View ArticleGoogle Scholar
- Christenson EM, Anseth KS, van den Beucken JJJP, Chan CK, Ercan B, Jansen JA, Laurencin CT: Nanobiomaterial Applications in Orthopedics. Journal of Orthopaedic Research. 2006, 25 (1): 11-22. 10.1002/jor.20305.View ArticleGoogle Scholar
- Sanosh KP, Chu MC, Balakrishnan A, Kim TN, Cho SJ: Utilization of biowaste eggshells to synthesize nanocrystalline hydroxyapatite powders. Materials Letters. 2009, 63: 2100-2102. 10.1016/j.matlet.2009.06.062.View ArticleGoogle Scholar
- Tsai WT, Yang JM, Hsu HC, Lin CM, Lin KY, Chiu CH: Development and Characterization of mesoporosity in eggshell ground by planetary ball milling. Microporous and Mesoporous Materials. 2008, 111: 379-386. 10.1016/j.micromeso.2007.08.010.View ArticleGoogle Scholar
- Matsuya S, Lin X, Udoh KI, Nakagawa M, Shimogoryo R, Terada Y, Ishikawa K: Fabrication of porous low crystalline calcite block by carbonation of calcium hydroxide compact. J Mater Sci: Mater Med. 2007, 18: 1361-1367. 10.1007/s10856-007-0123-4.Google Scholar
- Bagambisa FB, Joos U, Schilli W: Mechanism and structure of the bond between bone and hydroxyapatite ceramics. J Biomed Mater Res. 1993, 27: 1047-1055. 10.1002/jbm.820270810.View ArticleGoogle Scholar
- Neo M, Nakamura T, Ohtsuki C, Kokubo T, Yamamuro T: Apatite formation of three kinds of bioactive materials at an early stage in vivo: A comparative study by transmission electron microscopy. J Biomed Mater Res. 1993, 27: 999-1006. 10.1002/jbm.820270805.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.